Evaluation of Inflammation and Oxidative Stress Markers in Patients with Obstructive Sleep Apnea (OSA)

Abstract: Background: The identification of circulating markers of oxidative stress and systemic inflammation might enhance risk stratification in obstructive sleep apnea (OSA). We investigated the association between specific haematological parameters, as easily measurable markers of oxidative stress and inflammation, and the degree of hypoxia during polysomnography using the apnea hypopnea index (AHI), oxygen desaturation index (ODI), and oxygen saturation (SpO2), in OSA patients. Methods: Associations between polysomnographic parameters and demographic, clinical, and laboratory characteristics were assessed in a consecutive series of patients with OSA attending the Respiratory Disease Unit of the University Hospital of Sassari, north Sardinia (Italy), between 2015 and 2019. Results: In 259 OSA patients (195 males and 64 females), the body mass index (BMI) was significantly and positively associated with the AHI and ODI, and negatively associated with the mean SpO2. No haematological parameter was independently associated with the AHI or ODI. By contrast, albumin, neutrophil, and monocyte counts, and the systemic inflammatory response index (SIRI) were independently associated with a lower SpO2. Conclusions: Our results suggest that albumin and specific haematological parameters are promising markers of reduced oxygen saturation in OSA.


Introduction
Obstructive sleep apnea (OSA) is a leading public health issue affecting over 900 million people worldwide [1,2]. In Italy, the most recent epidemiological data published by the Ministry of Health estimate that moderate-severe and mild-moderate OSA affect 27% and 54% of the adult population, respectively [3].
OSA is characterised by intermittent and repeated episodes of upper airway collapse during sleep, resulting in partial (hypopnea) or complete (apnea) airflow obstruction [4]. This causes excessive daytime sleepiness with frequent awakenings, characterised by repeated episodes of intermittent hypoxia followed by rapid reoxygenation [5]. Patients with OSA are known to have a high risk of developing ischemic heart disease, heart failure, arrhythmia, stroke, and transient ischemic attack [6,7].
The cycles of deoxygenation and re-oxygenation caused by intermittent hypoxemia in patients with OSA have been shown to induce an excessive production of reactive oxygen species (ROS), which causes structural and functional damage to proteins, DNA, and lipids [8,9]. In addition, intermittent hypoxemia is associated with the production of pro-inflammatory factors, metabolic dysregulation, and platelet aggregation [10].
Although the exact mechanisms involved are not fully understood, several studies suggest that, in OSA patients, oxidative stress and systemic inflammation play a key pathophysiological role in the onset and progression of cardiovascular morbidity and vascular dysfunction [11,12].
The gold standard for the diagnosis of OSA, polysomnography (PSG), allows the systematic collection of various parameters simultaneously during sleep. However, it requires access to specialised centres and equipment [13].
Therefore, several investigators have proposed the use of haematologically based inflammatory and oxidative stress parameters as easily measurable markers for the early diagnosis and monitoring of OSA [14,15]. Tumour necrosis factor (TNF), C-reactive protein (CRP), and interleukins-6 (IL-6) represent the most studied inflammatory markers in OSA, and their increase has been observed in OSA, patients compared to controls [16]. Additionally, the use of alternative hematological parameters present in the blood count as specific biochemical markers of the disease has recently gained increasing interest [15]. Such parameters, e.g., the neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), and monocyte to lymphocyte ratio (MLR), have been investigated in several disease states [17][18][19][20][21]. A recent meta-analysis reported that the NLR could be a reliable marker to evaluate systemic inflammation and predict the severity of disease in OSA patients [22]. Among the markers of oxidative stress, albumin is the most abundant circulating protein that possesses significant antioxidant activity and is routinely measured [23]. A reduction of the antioxidant properties of serum albumin has been shown to be significantly associated with the presence and severity of OSA [24].
The apnea-hypopnea index (AHI), which indicates the number of apneas or hypopneas recorded per hour of sleep, is commonly used to diagnose OSA and determine its severity [25]. However, the AHI does not consider the duration of respiratory cessations and subsequent intermittent oxygen desaturations, which seem to play an important role in the development of complications related to OSA [26]. Recently, alternative parameters such as oxygen saturation (SpO 2 ), percentage of time with SpO 2 below 90%, and oxygen desaturation index (ODI), have also been used to assess the severity of OSA [27][28][29]. In this context, the aim of our study was to examine the relation between specific haematological parameters as markers of oxidative and inflammatory stress and the degree of hypoxia, measured by AHI, ODI, and SpO 2 , in a cohort of OSA patients in order to identify promising biomarkers of the disease.

Study Population
We conducted a retrospective study of a consecutive series of patients with OSA, diagnosed by polysomnography (PSG), attending the Respiratory Disease Unit of the University Hospital of Sassari, north Sardinia (Italy), between 2015 and 2019. Patients with AHI < 5 events/h, central sleep apnea, and receiving treatment were excluded.
Finally, AHI, ODI, and SpO 2 mean values during PSG were collected from each patient. Apnea events were defined as the absence, or almost total absence, of oro-nasal airflow for a period ≥ 10 s. Hypopnea was defined as respiratory events by an airflow reduction of ≤50% of baseline for ≥10 s [25]. The AHI indicates the average number of apnea and hypopnea episodes per hour of sleep, while the ODI measures the number of desaturation events per hour, where desaturation events represent a decrease in the mean oxygen saturation of ≥3% for at least 10 s. Patients were classified into 3 separate groups based on their AHI scores: mild (5 ≤ AHI < 15), moderate (15 ≤ AHI < 30), and severe OSA (AHI ≥ 30) [3].

Statistical Analysis
Data are expressed as means (mean ± SD) or medians (median and IQR). The variable distribution was assessed using the Kolmogorov-Smirnov test. Correlations between variables were assessed using Spearman's or Pearson's correlation. Multiple linear regression analysis was used to assess the presence of independent associations between AHI, ODI, and SpO 2 mean and clinical (BMI, smoking status, comorbidities, pharmacological treatment), demographic (age, sex), and laboratory variables (total and LDL cholesterol, ALT, AST, WBC, monocytes, lymphocytes, neutrophils, platelets, HDW, RDW, MPV, albumin, NLR, PLR, MLR, SII, SIRI, and AISI) by correcting for confounders with a p < 0.05 in univariate analysis. Non-normally distributed variables were log10-transformed prior to analysis using parametric tests. To avoid collinearity bias, the independent association of neutrophils, monocytes, NLR, PLR, MLR, SII, SIRI, and AISI with AHI, ODI, and SpO 2 was assessed in separate models. Statistical analyses were performed using MedCalc for Windows, version 20.109-64 bit (MedCalc Software, Ostend, Belgium).
Significant positive relationships were observed between AHI and ODI and the following laboratory parameters: HDW (AHI: r = 0.13, p = 0.028; ODI: r = 0.14, p = 0. In multivariate regression analysis, the AHI was independently associated with the BMI in all the models studied (Table 3). The ODI was independently correlated with BMI, hypercholesterolemia, and metformin (Table 4). While no association was observed between AHI or ODI and inflammatory parameters, particularly NLR, SII, SIRI, and AISI, that SpO 2 was strongly and independently associated with BMI, albumin, and specific inflammatory parameters, e.g., neutrophils, monocytes, and SIRI (Table 5).

Discussion
OSA is known to be associated with different cardiovascular, metabolic, and neurodegenerative disease states [31]. In this condition, oxidative stress and inflammation, induced by intermittent hypoxia, seem to play a key pathophysiological role in OSA [32]. Several blood cell components and combined cell count indexes are increasingly being studied as markers of inflammation in several inflammatory disorders, including OSA [17,19,32,33]. However, only a few reports have evaluated the prognostic capacity of these indexes, primarily NLR and PLR, with conflicting results [18,22]. The investigation of the systemic inflammatory response index (SII) in OSA was performed only in two studies reporting a significant positive correlation between AHI and SII [34,35].
We sought to address this issue by investigating promising haematological parameters associated with intermittent hypoxia in OSA patients, including the combined indices of inflammation (NLR, MLR, PLR, SII, SIRI, and AISI). In fact, it has been shown that these indices provide more information about the presence of an inflammatory state when compared with individual blood cell types [36]. Multivariate analysis showed no significant associations between measures of disease severity, AHI, and simple or combined indices, although NLR and SII were associated with AHI in univariate correlation analysis, in accordance with previous studies [34,35].
Considering the current interest in alternative parameters to assess the severity of OSA [25,26], we also investigated the association between the inflammatory parameters and the grade of hypoxia, using the ODI and oxygen saturation.
The ODI was not independently associated with any blood parameter. The SpO 2 mean exhibited a significant negative correlation with neutrophil and monocyte counts and with all the combined indexes barring PLR (p = 0.94). In multivariate analysis, neutrophil and monocyte counts and SIRI were independently associated with SpO 2 mean reduction.
Chronic intermittent hypoxia and sleep fragmentation interact variably with the immune system, which triggers pro-inflammatory pathways and cellular activation [37]. In this report, the cells most affected by oxygen saturation appear to be neutrophils and monocytes. The neutrophils possess the ability to produce ROS under hypoxic conditions with an increase in recruitment, stimulation of degranulation, and survival of neutrophils through several pathways, including the NF-κB binding of hypoxia-inducible factor (HIF−1), the main regulator of oxygen homeostasis [38,39]. Additionally, it has been observed that the induced hypoxic stress is associated with an increase in monocytes and with the activation of the production of inflammatory mediators by these cells [40].
In our study, the SIRI parameter, which combines neutrophils and monocytes in relation to lymphocytes, further confirmed the presence of a significant association between alterations in these cells and a reduction of the mean saturation.
We also demonstrated that mean SpO 2 was strongly and independently associated with serum albumin in all models examined, whereas no correlation between AHI and ODI with albumin was found. Serum albumin is an important antioxidant agent, and structural changes caused by free radicals interfere with its antioxidant properties. In OSA, it has been reported that the reduction of antioxidant capacity and the consequent aggravation of oxidative stress can also contribute to cardiovascular and metabolic abnormalities [24]. Additionally, it has been observed that ischemia-modified albumin (IMA), created by the modification of albumin by free radicals, increased significantly in severe OSA patients, and its elevation seems to be reversed by CPAP treatment [41].
Our results showed that SpO 2 can be mainly related to the reduction of antioxidant properties induced by intermittent hypoxia when compared with the index of severity of AHI. In this context, studies on the evaluation of the association between IMA and the degree of hypoxia could be useful to further confirm our data.
Finally, we confirmed data previously obtained in other studies, showing that the severity of OSA and oxygen saturation are modulated by weight changes [42,43]. In fact, a significant positive relationship between AHI, ODI, and BMI was observed, and a higher BMI strongly correlated with a lower SpO 2 mean. These results further highlight that the presence of obesity results in more severe episodes of obstruction and desaturation events and, consequently, lower oxygen saturation during polysomnography [44].
Our study has some limitations, such as the retrospective design, the relatively small sample size, and the lack of comparison with control subjects.
Nevertheless, this is the first study evaluating the role of SIRI, AISI, and albumin in OSA patients in correlation with different PSG parameters, particularly oxygen saturation. Moreover, it could be a reference study for future case-control research, which could better clarify the utility of the parameters analysed in the OSA condition.
In conclusion, these results suggest that lower oxygen saturation strongly correlates with a reduction in the antioxidant properties of albumin and a pro-inflammatory profile characterised by an increased number of monocytes and neutrophils and a higher systemic inflammatory response index (SIRI). Pending the results of larger prospective studies including control subjects, these findings suggest a possible role for oxygen saturation in assessing the inflammation and oxidative stress burden in OSA.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee of the University Hospital (AOU) of Cagliari (PG/2022/6361).